全反式维甲酸联合射线对食管癌细胞TE13增殖及凋亡的影响

目的:研究全反式维甲酸( all-trans-retinoicacid,ATRA)联合放射线照射对食管癌TE13细胞增殖、凋亡的影响及可能的作用机制.方法:采用MTT法检测ATRA对人食管癌细胞TE13增殖的影响；以其对TE 13细胞生长抑制率(inhibitory rate,IR)达25％、50％和75％时的ATRA的浓度分别联合γ-射线进行分组处理细胞；FCM法分析ATRA单药及联合放射治疗对TE13细胞周期及凋亡的影响；采用克隆形成实验分析对克隆形成率和细胞存活率的影响；采用FCM法检测cyclinD1蛋白的表达.结果:ATRA对TE13细胞有增殖抑制作用,且呈剂量和时间依赖性；ATRA浓度为0.78、6.25和12.5 μmol/L作用48 h后,其对TE13细胞的IR分别达到22.0％、55.1％和71.1％.ATRA联合γ-射线(4 Gy)与ATRA单药组比较,可明显降低TE13细胞的存活率和克隆形成率；ATRA联合γ-射线(4 Gy)处理TE13细胞24和48 h后,细胞增殖能力明显下降,更多的细胞被阻滞于G0/G1期,细胞凋亡率明显增加,与ATRA单药组比较,联合放射治疗可明显增强对TE13细胞周期和凋亡的影响；ATRA浓度为0.78 μmol/L时和γ -射线联合作用对细胞周期cyclinD1蛋白表达量影响不大,但ATRA浓度为6.25和12.5 μmol/L时则能明显下调cyclinD1的表达.结论:ATRA对TE 13细胞具有明显的增殖抑制作用,其可能通过下调cyclinD1蛋白的表达,将TE13细胞阻断于G0/G1期并诱导细胞凋亡.在ATRA浓度较高时联合放疗,能显著下调cyclinD1蛋白表达,加强细胞G0/G1期阻滞及促进凋亡,抑制细胞克隆的形成能力；在ATRA浓度较低时联合放疗对原代细胞cyclinD1蛋白表达量的影响较小,但对促进细胞凋亡、抑制细胞克隆仍有一定的效果,其损伤效应可能需要一定时间.

Effects of all-trans retinoic acid combined with gamma radiation on proliferation and apoptosis of esophageal carcinoma TE13 cells

Objective:To investigate the effects of ATRA (all-trans-retinoicacid) combined with gamma radiation on proliferation and apoptosis of human esophageal carcinoma TE13 cells, and to explore the possible mechanism. Methods:The effect of ATRA on the proliferation of TE13 cells was detected by MTT method. When the cell growth RI (inhibitory rate) reached levels of 25%, 50% and 75%, the TE13 cells were treated with the corresponding inhibitory doses of ATRA combined with 4 Gy gamma radiation. The effects of this combination intervention on cell cycle distribution and apoptosis of TE13 cells were detected by FCM (flow cytometry). The colony-formation ability and cell viability were detected using colony-formation experiment. The expression of cyclinD1 protein was detected by FCM. Results:The inhibitory effect of ATRA on the proliferation of TE13 cells was significant in a dose- and time-dependent manner. The cell growth IRs reached 22.0%, 55.1% and 71.1% at ATRA concentrations of 0.78, 6.25 and 12.5 μmol/L, respectively. The cell viability and colony-formation efficiency were significantly decreased in TE13 cells treated with ATRA in combination with 4 Gy gamma radiation, as compared with TE13 cells receiving administration of ATRA alone. The proliferative ability of TE13 cells was significantly reduced after ATRA treatment in combination with 4 Gy gamma radiation for 24 and 48 h; furthermore, the percentage of the cells arrested at phase G0/G1 was increased accompanying with a significantly elevated apoptotic rate. Although the combination treatment (0.78 μmol/L ATRA and gamma radiation) had a weak influence on the expression of cyclinD1 protein, which was significantly decreased in other groups (6.25 and 12.5 μmol/L ATRA). Conclusion:ATRA exerts an inhibitory influence on the proliferation of TE13 cells through down-regulating cyclinD1 expression, arresting the cells at phase G0/G1, and inducing apoptosis. A higher-concentration of ATRA combined with gamma radiation can significantly decrease the expression of cyclinD1, promote G0/G1 cell cycle arrest, accelerate apoptosis, and limit the colony formation, but a lower concentration of ATRA combined with gamma radiation exerts a little influence on cyclinD1 expression, although it may accelerate apoptosis and limit the colony-formation at some periods of time after treatment.